Spectroscopic properties of chymotrypsin and model compounds indicate that a low-barrier hydrogen bond participates in the mechanism of serine protease action. A low-barrier hydrogen bond between Nδ1 of His 57 and the β-carboxyl group of Asp 102 in chymotrypsin can facilitate the formation of the tetrahedral adduct, and the nuclear magnetic resonance properties of this proton indicate that it is a low-barrier hydrogen bond. These conclusions are supported by the chemical shift of this proton, the deuterium isotope effect on the chemical shift, and the properties of hydrogen-bonded model compounds in organic solvents, including the hydrogen bond in cis -urocanic acid, in which the imidazole ring is internally hydrogen-bonded to the carboxyl group.